2.1 Isolation From Natural Sources and Therapeutic Utility of Indolylquinones
Research interest concerning indolylquinones grew out of early observations that extracts of Chaetomium exhibited antibiotic properties. These observations led researchers to attempt the isolation of active species from cultures of these microorganisms. For example, Brewer et al. disclose the isolation of a purple pigment, which was termed cochliodinol, from isolates of Chaetomium cochliodes and Chaetomium globosum (1968, "The Production of Cochliodinol and a Related Metabolite by Chaetomium Species," Can. J. Microbiol. 14:861-866). Brewer et al. also disclose the synthetic conversion of cochliodinol to a diacetate compound. Id. Further, the antifungal properties of cochliodinol have also been documented (Meiler et al., 1971, "The Effect of Cochliodinol, a Metabolite of Chaetomium cochliodes on the Respiration of Microspores of Fusarium oxysporum," Can. J. Microbiol. 17: 83-86).
The structure of cochliodinol was elucidated by Jerram et al. in 1975. (1975, "The Chemistry of Cochliodinol, a Metabolite of Chaetomium spp.," Can. J. Chem. 53:727-737). Jerram et al. reported the structure of cochliodinol as: 2,5-dihydroxy-3,6-di(5'-(2"-methylbut-.DELTA..sup.2 "-ene)-indolyl-3')-cyclohexadiene-1,4-dione. The conversion of cochliodinol to various other derivatives, including its dimethyl and diacetyl analogues, was also disclosed. Id. Some of these derivatives were highly colored and suitable for use as dyes, while others were colorless. Id. Sekita discloses the isolation of other bis(3-indolyl)-dihydroxybenzoquinones, including isocochliodinol and neocochliodinol from Chaetomium muroum and C. amygdalisporum (1983, "Isocochliodinol and Neocochliodinol, Bis(indolyl)-benzoquinones from Chaetomium spp.," Chem. Pharm. Bull. 31(9): 2998-3001).
Despite the therapeutic potential of cochliodinol and its derivatives, efficient methods suitable for large scale production of these compounds have remained elusive. U.S. Pat. No. 3,917,820 to Brewer et al. discloses the purple pigment cochliodinol and a process for its production by culturing various types of Chaetomium under aerobic conditions. However, the methods of Brewer require long incubation periods for cochliodinol production (2-8 days), the use of benzene, a known carcinogen, to effect chromatographic separation of cochliodinol from the culture and are limited to the few naturally occurring compounds. Moreover, Brewer discloses the isolation of only small quantities (0.75 grams) of cochliodinol from Chaetomium.
Another class of indolylquinones known as the asterriquinones in which the nitrogen of the indole ring is substituted, has been shown to exhibit antitumor activity. Arai et al. proposed the general name "asterriquinones" for the class of indolylquinones based upon asterriquinone (1981, "Metabolic Products of Aspergillus terreus IV. Metabolites of the Strain IFO 8835. (2) The Isolation and Chemical Structure of Indolyl Benzoquinone Pigments," Chem. Pharm. Bull. 29(4): 961-969). It should be noted that as used herein, the term "asterriquinone" has a more general meaning, and is used interchangeably with the term "indolylquinone." Yamamoto et.al. disclose the antitumor activity of asterriquinone, i.e., 2,5-bis[N-(1",1"-dimethyl-2"-propenyl)indol-3'-yl]-3,6-dihydroxy-1,4-benzo quinone, and its isolation from the fungus Aspergillus terreus (1976, "Antitumor Activity of Asterriquinone, a Metabolic Product from Aspergillus terreus," Gann 67:623-624).
Arai et al. disclose the isolation and characterization of 11 different kinds of bisindolyl-dimethoxyl-p-benzoquinones from Aspergillus terreus. Id. The isolation and structural determination of a number of other asterriquinones have also been reported. (Arai et al. 1981, "Metabolic Products of Aspergillus terreus VI. Metabolites of the Strain IFO 8835. (3) the Isolation and Chemical Structures of Colorless Metabolites," Chem. Pharm. Bull. 29(4): 1005-1012; Kaji et al., 1994, "Four New Metabolites of Aspergillus Terreus", Chem. Pharm. Bull. 42(8): 1682-1684). However, the separation of asterriquinones is troublesome because there are so many kinds of homologous pigments in the Aspergillus extracts. Moreover, the chromatographic purification of asterriquinones is typically carried out using benzene, a known carcinogen, as a solvent. Finally, only milligram quantities of asterriquinones have actually been isolated from these natural sources.
In view of their potential as anticancer agents, research has been directed to determination of the relationship between structure and antitumor activity of asterriquinones. For example, Arai et al. reported a study in which hydroxyl benzoquinone derivatives obtained by demethylation of bisindolyl-dimethoxyl-p-benzoquinones were found to have greater antitumor activity than the methoxyl derivatives (1981, "Metabolic Products of Aspergillus terreus V. Demethylation of Asterriquinones," Chem. Pharm. Bull. 29(4): 991-999). Shimizu et al. noted that the presence of free hydroxyl groups in the benzoquinone moiety, as well the number and position of tert-, isopentenyl, or both pentyl groups, seems to have an effect on the antitumor activity of the compound (1982, "Antitumor Effect and Structure-Activity Relationship of Asterriquinone Analogs," Gann 73: 642-648). In an attempt to obtain information towards the development of more potent asterriquinone derivatives, Shimizu et al. conducted an investigation into the structure-activity relationship of asterriquinones in which the action mechanism of asterriquinone in its antitumor activity with reference to its interaction with DNA molecules and the plasma membrane of tumor cells was studied (1990, "Interaction of Asterriquinone with Deoxyribonucleic Acid in Vitro," Chem. Pharm. Bull. 38(9): 2617-2619). It was reported that a correlation exists between the pKa value of the asterriquinone derivative and its antitumor activity. Id. Maximum antitumor activity was observed for compounds with pKa's in the range of 6-7. Id.
Analysis of structure-activity relationships has led to attempts to obtain compounds with more potent antitumor activity by chemical modification of asterriquinone and related compounds isolated from natural sources (Shimizu et al., 1982, "Antitumor Activity of Asterriquinones from Aspergillus Fungil IV. An Attempt to Modify the Structure of Asterriquinones to Increase the Activity," Chem. Pharm. Bull. 30(5): 1896-1899). Although benzoquinone derivatives having aziridinyl groups in the molecule such as mitomycin C, carbazilquinone or "E 39" are well known potent anticancer agents, replacement of the functional groups at the 3 and 6 positions in the benzoquinone moiety of asterriquinone failed to enhance its antitumor potency. Id. Similarly, the introduction of an ethyleneimino group into the molecule did not increase antitumor activity. A dimethylallyl derivative of asterriquinone showed moderate activity against the ascites and solid tumors of Ehrlich carcinoma, while an allyl derivative did not. It was suggested that in order to enhance the antitumor activity, it may be necessary not only to alter the pKa value by alkylation, but also to introduce hydrophilic groups into the molecule.
Most recently, in addition to their demonstrated antitumor activity, asterriquinone and some of its analogues have also been shown to be strong inhibitors of HIV-reverse transcriptase (Ono et al., 1991, "Inhibition of HIV-Reverse Transcriptase Activity by Asterriquinone and its Analogues," Biochem. Biophys. Res. Commun. 174(1): 56-62).
2.2 Cancer and Signal Transduction
As mentioned above, indolylquinones have utility as antitumor agents for the treatment of cancer and other cell proliferative disorders. These compounds are believed to arrest the growth of tumors by interfering with the signal transduction pathways that regulate cell proliferation and differentiation.
Protein phosphorylation is a common regulatory mechanism used by cells to selectively modify proteins carrying signals that regulate cell proliferation and differentiation. The proteins that execute these biochemical modifications are a group of enzymes known as protein kinases. They may further be defined by the amino acid that they target for phosphorylation. One group of protein kinases are the tyrosine kinases (PTKs) which selectively phosphorylate a target protein on its tyrosine residues.
Protein tyrosine kinases comprise a large family of proteins, including many growth factor receptors and potential oncogenes. Tyrosine kinases can be cytoplasmic, non-receptor-type enzymes and act as a key component of a signal transduction pathway which regulates cell functions such as cell division, differentiation and survival.
Adaptor proteins are intracellular proteins having characteristic conserved peptide domains (SH2 and/or SH3 domains, as described below) which are critical to the signal transduction pathway. Such adaptor proteins serve to link protein tyrosine kinases, especially receptor-type protein tyrosine kinases to downstream intracellular signalling path-ways such as the RAS signalling pathway. It is thought that such adaptor proteins may be involved in targeting signal transduction proteins to the correct site in the plasma membrane or subcellular compartments, and may also be involved in the regulation of protein movement within the cell.
The profound cellular effects mediated by tyrosine kinases and adaptor molecules have made them attractive targets for the development of new therapeutic molecules. It is known, for example, that the overexpression of tyrosine kinases, such as HER2, can play a decisive role in the development of cancer (Slamon, D. J., et al. , 1987, Science, 235:177-182) and that antibodies capable of blocking the activity of this enzyme can abrogate tumor growth. (Drebin, et al. 1988, Oncogene 2:387-394). Blocking the signal transduction capability of tyrosine kinases such as Flk-1 and the PDGF receptor have been shown to block tumor growth in animal models (Millauer, B., et al. 1994, Nature 367:577; Ueno, H., et al. 1991, Science 252:844-848).
Despite great interest in the various therapeutic and other utilities of indolylquinones such as asterriquinones, research into the therapeutic activities of indolylquinones and efforts to obtain indolylquinones with enhanced therapeutic activity have both been limited by the lack of reliable sources for these compounds. Indeed, isolation of indolylquinones from natural sources requires multiple steps and produces only milligram quantities of the target molecules. Further, evaluation of the activities of novel indolylquinones has necessarily been confined to those compounds which can be obtained by chemical modification of known compounds that can be isolated from natural sources. Clearly, a synthetic routine to these compounds would be invaluable to the art.
2.3 Synthesis of Cochliodinol
A synthetic route to an indolylquinone, cochliodinol, has been reported by Horcher et al. This route is a complex, multi-step, low-yield process for the total synthesis of cochliodinol (1986, "Totalsynthese des Cochliodinols", Liebigs. Ann. Chem. 1765-1771). The Horcher method involves an unusual solid state reaction of bromanil (2,3,5,6-tetrabromo-1,4-quinone) with 5-bromoindole in the presence of aluminum oxide and potassium carbonate in a dry box at 105.degree. C. This solid state reaction yields about 11% of 2,5-dibromo-5,6-bis(5-bromo-3-indolyl)-1,4-quinone. The 2,5-dibromo-5,6-bis(5-bromo-3-indolyl)-1,4-quinone is then treated with benzalcohol and sodium hydroxide to give 2,5-bis(benzyloxy)-3,6-bis(5-bromo-3-indolyl)-1,4-quinone in 45% yield. This product is then reacted with hydrogen gas in the presence of a 10% Pd on activated charcoal catalyst, followed by treatment with acetic anhydride in pyridine to give 1,2,4,5-tetracetoxy-3,6-bis(5-bromo-3-indolyl)benzene. Reaction of this compound with a complex of isopentenyl bromide and tetracarbonyl nickel gives 1,2,4,5-tetracetoxy-3,6-bis[5-(3-methyl-2-butenyl)3-indolyl]benzene. This compound is then reacted with sodium hydroxide and oxygen to give cochliodinol.
According to Horcher et al., the reaction of bromanil with certain substituted indoles is problematic. Horcher et al. report that earlier attempts to react a p-benzoquinone with 2-methylindole resulted in only monoindolequinones in very low yields. Attempts to react bromanil with 5-(2-methylbut-2-en-4-yl)-indole were also reported by Horcher to be unsuccessful due to the instability of the unsaturated side chain vis-a-vis the dehydrogenating bromanil. To overcome this difficulty, Horcher reacted bromanil with 5-bromoindole instead of 5-(2-methylbut-2-en-4-yl), followed by introduction of the 2-methylbut-2-en-4-yl group at the end of the synthesis, requiring the additional step of reacting the 5,5'-dibromo-bis-indolylquinone with the complex of isopentenyl bromide and tetracarbonyl nickel, which substitutes the bromine atoms with 2-methylbut-2-en-4-yl groups.
Horcher et al. report that this method resulted in isolation of only milligram quantities of cochliodinol in a very low overall yield. However, Horcher et al. indicate that conducting the initial reaction of bromanil with 5-bromoindole in smaller batches results in better yields. This suggests that the methods of Horcher et al. are unsuited for production of bis-indolylquinones on a large scale. In addition, as applied to the production of bis-indolylquinones in general, the methods of Horcher et al. would be prohibitively multistep, and would likely result in isolation of only milligram quantities of the target indolylquinones. Moreover, these methods require high temperature and manipulation in dry box.
Accordingly, despite the great interest in indolylquinones, there is a lack of feasible large scale synthetic routes for obtaining these compounds. Thus, there is a need in the art for a fast, efficient synthetic method for making indolylquinones in preparative quantities. Further, there is a need for synthetic means of producing known indolylquinones previously available only in milligram quantities from natural sources. Moreover, there is a need in the art for a synthetic method that may be manipulated easily to produce a wide variety of structurally diverse novel indolylquinones, so that structure-activity relationships may be further elucidated, and new, perhaps more therapeutically useful indolylquinones may be developed.